Alzheimer's disease (AD) is nearing an epidemic due to a medical paradox: as the general health and lifespan of the population increases, diseases of aging increase in prevalence. Age correlates strongly with AD, affecting 1 in 8 over 65 and nearly 1 in 2 over 85. The startling increase in the number of affected individuals is straining healthcare, particularly as the Baby Boomer generation ages into their 60s and beyond. Yet, few drugs are currently available for treatment or alleviation of AD. The rational design of effective therapeutics can be more efficiently realized with a thorough understanding of the mechanism by which AD manifests itself. At a genetic and cellular level, the roles of the protease gamma-secretase and its substrate, amyloid precursor protein (APP), in the etiology of AD have been known for years. Yet at the molecular level, virtually nothing has been described for the mechanism by which gamma-secretase cleaves APP to give the neurotoxic A-beta peptides. This project specifically addresses three independent aims: 1. The current methods for measuring gamma-secretase activity will be optimized. Various metals, cofactors, and lipids will be screened for their mechanism of modulating gamma-secretase cleavage of substrates in vitro. An existing assay using purified, reconstituted gamma-secretase will be optimized by reconstituting gamma-secretase and its substrates within the same liposome or vesicle. Finally, design of a fluorescence-based assay will allow greater sensitivity and more rapid analysis of activity. 2. The rates and mechanism by which gamma-secretase cleaves its substrates will be elucidated. In vitro assays will be used to determine Km and kcat for various substrates and familial Alzheimer's disease mutants and to characterize inhibitors. Moreover, single-turnover experiments and pre-steady state kinetic methods will be used to elucidate the kinetics and mechanism of each step of gamma-secretase activity. 3. The mechanism by which substrates are differentially recognized and cleaved will be elucidated. Using mutants and binding analysis, the apparent promiscuity of gamma-secretase cleavage will be clarified. Relevance: gamma-secretase is the protein responsible for forming the deadly amyloid-beta plaques found in the brains of Alzheimer's disease patients. This research proposal aims to dissect the individual molecular steps of gamma-secretase activity and the rates at which these steps occur. Such a detailed analysis of the process by which gamma-secretase acts is crucial to the rational design of drugs to alleviate, treat, or ultimately cure Alzheimer's disease.